Wound spray

ABSTRACT

The present invention refers to a composition, comprising hemoglobin or myoglobin, wherein in at least 40% of said hemoglobin or myoglobin the oxygen binding site is charged by a non-O 2  ligand, and at least one further ingredient, a method for preparing said composition and the use of hemoglobin or myoglobin charged with a non-oxygen ligand for external treatment of wounds.

The present invention refers to a composition, comprising hemoglobin ormyoglobin, wherein in at least 40% of said hemoglobin or myoglobin theoxygen binding site is charged by a non-O₂ ligand, and at least onefurther ingredient, a method for preparing said composition and the useof hemoglobin or myoglobin charged with a non-oxygen ligand for externaltreatment of wounds.

Different methods are used for treating wounds, depending on theirstatus. First, a wound that is still open preferably should bedisinfected and thereby protected against negative external influences.This can be done by means of suitable disinfectant solutions or spray-onbandages or also by applying iodine solution. Actual wound healing mustthen take place from inside. This means that the blood vessels still inplace must supply the destroyed tissue with sufficient amounts ofsubstrates, so that the tissue repair mechanism can start.

Wounds can be caused by various factors, like e.g. injuries or alsoafter operations or traumatic events.

On the other hand, it is known that wound formation, particularly alsochronic wounds, can also be provoked by diseases, in which degenerationand/or constriction of large and/or small blood vessels occurs. This maybe the result e.g. in the case of older patients, of extended stays inbed (decubitus) or of diabetes mellitus which may lead to degenerationand arteriosclerosis (P. Carpenter, A. Franco, Atlas der Kapillaroskopie[Atlas of Capillaroscopy], 1983, Abbott, Max-Planck-Inst. 2,D-Wiesbaden) of the large and small blood vessels (macroangiopathy andmicroangiopathy of the arteries). It was also shown that an oxygendeficiency (hypoxia) is present in the wound area. 40 mmHg is consideredto be a critical value (C. D. Müller et al., Hartmann Wund [Wound] Forum1 (1999), 17-25).

The blood flows to the tissues, including the skin, through the arteriesand supplies the cells with substrates required for life. Anydegeneration of the blood vessels results in a deficient supply ofsubstrates to the cells, leading to their death. The substrates mustpass the last, seemingly insignificant gap of approximately 20 μm fromthe smallest blood vessels (capillaries) to the cells by diffusion; inthis connection, oxygen plays a special role, because it is difficultfor the organism to handle this substrate.

There are three main problems involved:

(1) It is true that oxygen is absolutely essential for life (a humanbeing is brain-dead after only approximately five minutes if his/herbrain does not receive oxygen), but at the same time, oxygen is highlytoxic (a newborn that receives respiration treatment with pure oxygenwill die-after only a few days).

(2) Oxygen has very little solubility in an aqueous medium. Thisresults, according to FICK's first law, in a lower diffusion rate ofoxygen. In addition, there is a fundamental law of diffusion, namelySMOLUCHOWSKI's and EINSTEIN's law, that states that the diffusion speed(of oxygen) decreases with an increasing diffusion distance. Now thediffusion constant of oxygen is so low that the diffusion speed at adiffusion distance of as little as 20 μm is only 5% of the initialvalue. A water layer of e.g. 50 μm represents a nearly complete oxygeninsulation for the cells. Oxygen is transported along the long paths inthe organism from the lungs to the tips of the toes with thebloodstream, bound to hemoglobin, and only in this way is able toovercome the long distances in a manner that is suitable for theorganism.

(3) For oxygen, in contrast to glucose, for example, there is no storagearea in the body, therefore this substrate must be available to thecells at all times and quickly, in a sufficient amount; oxygen is aso-called immediate substrate necessary for life.

An intact organism has solved these problems by using severalmechanisms. The toxic effects of oxygen are avoided in that the latterbinds during transport to hemoglobin and thereby remains harmless. Atthe same time, the free oxygen is diluted and thereby further loses itsharmful oxidative potential. Nevertheless, it is instantaneouslyavailable in a sufficient amount, because the binding to hemoglobin isreversible. The problem of the low diffusive range is solved in that theorganism has developed a very finely branched blood vessel network(capillary network), which ensures that on the average, every cell is ata distance of at most 25 μm from a capillary; in this way, the diffusionpath of oxygen in the organism remains below the critical length of 50μm. In addition, a cell can be diffusively supplied with oxygen fromseveral sides; this represents a safety mechanism. The immediateavailability, in accordance with the demand (oxygen is not allowed to beavailable in excess, otherwise it would have a harmful effect) isachieved, in the organism, by means of vascular regulation of the bloodvessel flow, which controls perfusion and thereby optimizes the supplyof oxygen.

If there is an open wound surface, the oxygen supply to the cells isinterrupted. The oxygen supply from air outside is poor because anaqueous liquid film is laying on the (tissue) cell layer, which film, asexplained, forms a diffusive oxygen barrier. Fresh wounds in normaltissue can heal in a few days, if the oxygen supply from underneath, inother words from the inside, is sufficient. However, it was shown inanimal experiments that even fresh wounds heal better if the oxygenconcentration of the surrounding air is increased (M. P. Pai et al.,Sug. Gyn. Obstet. 135 (1972), 756-758). Older, particularly chronicwounds are known to heal very slowly, if at all, due to their oxygendeficiency.

To heal chronic wounds better, as well, so-called hyperbaric oxygentherapy (HBO) has been used. In this treatment, patients are placed inpressurized chambers, where they are subjected to an excess pressure ofpure oxygen of about 3 bar for a certain period of time (C. D. Müller etal., Hartmann Wund Forum 1 (1999), 17-25). In fact, wound healing may beincreased by this method. However, the effect decreases with the numberof treatments.

U.S. Pat. No. 2,527,210 describes a hemoglobin solution that canallegedly be used for the treatment of wounds, both intravenously andtopically, for example by spraying. In this description, the hemoglobinis obtained from fresh erythrocytes that are subjected to freezing shockafter centrifugation and drawing off the blood plasma fraction. Thisresults in cell lysis, and hemoglobin is released. The broken cell wallsare also present in the product. This formulation is a concentrated celldetritus (cell fragments). In this way, an antiseptic cover effect suchas otherwise achieved with iodine solution, after having added 5% sodiumsulfide, is supposed. In other words, the wound is merely closed. Oxygentransport is not mentioned there.

WO 97/15313 describes the therapeutic use of hemoglobin for improvingwound healing. For this purpose, hemoglobin free of stroma and pyrogensis intravenously administered to the patients, particularly afteroperations and traumatic events to increase the blood pressure. Inparticular, a hemoglobin cross-linked with diaspirin is used for thispurpose.

WO 2003/077941 teaches the treatment of open wounds with a hemoglobinsolution comprising isolated and optionally crosslinked hemoglobin. Thesolutions were freshly prepared with hemoglobin from pig blood andapplied to chronic wounds.

During the preparation and storage of oxygen carriers on basis ofhemoglobin or myoglobin they can lose their functionality partially orcompletely. To prevent this it is desirable to stabilize the oxygencarriers that they remain usable and able to transport oxygen.

Generally, there are different approaches to the preparation ofartificial oxygen carriers; one of them is the preparation of suitablesolutions of native or chemically modified hemoglobins (see “Issues fromVth International Symposium on Blood Substitutes, San Diego, Calif.,USA, March 1993”, Artificial Cells, Blood Substitutes, andImmobilization Biotechnology 22 (1994), vol. 2-vol. 4). One problem inthe handling of such pharmaceutical preparations as artificial oxygencarriers is their increasing inactivation by spontaneous oxidation tomethemoglobin which is no longer able to transport oxygen. This occursusually during preparation by the producer and the subsequent storage.

Several approaches for solving this problem are described. Either it istried to minimize the degree of oxidation of hemoglobin, or to reducethe oxidized hemoglobin back again.

One possibility for prevention of spontaneous oxidation is deoxygenatingthe hemoglobin (i.e., entirely removing oxygen from the preparation),since desoxyhemoglobin oxidizes much more slowly to methemoglobin thanoxyhemoglobin.

Further it is possible to minimize the amount of oxidation by storageand/or preparation at the lowest possible temperature (for aqueoussolutions, at about 4° C.).

Additionally, the rate of oxidation of hemoglobin depends on thehydrogen ion concentration, i.e., the pH. For example, for native humanhemoglobin there is a minimum in the interval between pH 7.5 and 9.5.

Also, the addition of certain alcohols can diminish the oxidation ofhemoglobin. Some of them work even in low concentration. One problem isthe toxicity of these alcohols.

Certain metal ions (Cu₂ ⁺, Fe₃ ⁺ etc.) catalyze the spontaneousoxidation of hemoglobin. They can be rendered ineffective by complexingwith EDTA (ethylenediaminetetraacetic acid), although EDTA itselfpromotes the spontaneous oxidation of hemoglobin.

Protection of artificial oxygen carriers against oxidation may furtherbe achieved by the addition of reducing substances. Under certaincircumstances they even result in a reactivation of oxidized hemoglobin.

EP 0 857 733 describes that hemoglobin may be stabilized by binding aligand, in particular carbon monoxide, to the oxygen binding site. Itwas found that such a carbonylhemoglobin can be applied to an organismwithout de-ligandation and is suitable as an oxygen carrier inside ofthe blood stream.

The object of the present invention is to provide a product for theexternal treatment of wounds, which increases the wound healing, is easyin handling and storable.

This object is met, according to the invention, by a composition,comprising

-   -   (a) An oxygen carrier, preferably hemoglobin or myoglobin,        wherein in at least 40% of said oxygen carrier the oxygen        binding site is charged by a non-O₂ ligand, and    -   (b) at least one further ingredient, selected from        electrolyte(s) preservative(s), stabilizer(s),        anti-flocculant(s), anticoagulant(s), pH buffering agent(s),        solvent(s), antioxidant(s), film-forming agent(s) and        crosslinking agent(s)        and the use of an oxygen carrier, preferably hemoglobin or        myoglobin charged on its oxygen-binding site with a non-O₂        ligand for the preparation of an agent or composition for the        external treatment of wounds.

According to the invention preferably a natural (native) oxygen carrier,particularly hemoglobin or myoglobin or a modified derivative thereof,or mixtures thereof, is/are used. Hemoglobin or myoglobin of human oranimal origin, in particular of equine, bovine or preferably porcineorigin, is particularly suitable for the present invention. Human orporcine hemoglobin, which is natural or modified as described below, isparticularly preferred as an oxygen carrier. The oxygen carrier may befreshly isolated from human or animal blood or may be artificiallyprepared.

Mixtures of natural and modified oxygen carrier can also be used, suchas, for example, in a ratio of 20:1 to 1:20, with reference to weight.Further, mixtures of hemoglobin and myoglobin, or their modifiedderivatives may be used in the aforementioned ratio of 20:1 to 1:20.

In a further embodiment the oxygen carrier may be modified. Themodification can be an intramolecular cross-linking, polymerization(intermolecular cross-linking), pegylation (covalent linking withpolyalkylene oxides), modification with chemically reactive effectorssuch as pyridoxal-5′-phosphate or 2-nor-2-formyl-pyridoxal-5′-phosphate,or also with chemically non-reactive effectors of the oxygen bond, suchas 2,3-bisphosphoglycerate, inositol hexaphosphate, inositolhexasulfate, or mellitic acid, or a combination thereof. Suchmodifications are known and described, for example, in DE-A 100 31 744,DE-A 100 31 742, and DE-A 100 31 740. Cross-linking of oxygen carriersis also described in DE 197 01 37, EP 97 1000790, DE 44 18 937, DE 38 41105, DE 37 14 351, DE 35 76 651.

Examples for modified oxygen carriers are hemoglobins having a molecularweight of 65,000 to 15,000,000, such as intramolecularly cross-linkedmolecules according to WO 97/15313, particularly polymer products aswell as intermolecularly cross-linked products having an averagemolecular weight of 80,000 to 10,000,000 g/mol, particularly 100,000 to5,000,000, or analogously produced myoglobins having a molecular weightof 16,000 to 5,000,000, particularly 100,000 to 3,000,000, preferably1,000,000 g/mol. Those oxygen carriers that are polymerized, for exampleusing cross-linking agents known for intermolecular modification, suchas bifunctional cross-linking agents like butadiene diepoxy, divinylsulfone, diisocyanate, particularly hexamethylene diisocyanate,cyclohexyl diisocyanate, and 2,5-bisisocyanatobenzol sulfonic acid,di-N-hydroxy succinimidyl ester, diimidoester, or dialdehyde,particularly glyoxal, glycol aldehyde that reacts analogously, orglutardialdehyde may be used.

Furthermore, products which are polymerized in this manner and pegylatedwith a polyethylene glycol or suitable derivatives thereof may be used.This includes, for example, polyethylene oxide, polypropylene oxide, ora copolymer of ethylene oxide and propylene oxide, or an ester, ether,or ester amide thereof. It may be suitable if the covalently linkedpolyalkylene oxide has a molar mass of 200 to 5000 g/mol.

For covalent linking of the polyalkylene oxides, those derivatives ofpolyalkylene oxide that contain a linking agent already covalently boundwith a functional group, thereby allowing a direct chemical reactionwith amino, alcohol, or sulfhydryl groups of the hemoglobins, formingcovalent links of the polyalkylene oxides may be suitable, for examplepolyalkylene oxides with reactive N-hydroxy succinimidyl ester, epoxy(glycidyl ether), ldehyde, isocyanate, vinyl sulfone, iodacetamide,imidazolyl formate, tresylate groups, and others. Many suchmonofunctionally activated polyethylene glycols are commerciallyavailable.

If modified oxygen carriers are used, modified cross-linked(intramolecular or intermolecular), or cross-linked and pegylatedhemoglobin products having an average molecular weight of 250,000 to750,000 g/mol, or myoglobin products having an average molecular weightof 50,000 to 750,000 g/mol, are preferred.

According to the particular preferred embodiment freshly isolatedhemoglobin or myoglobin of human or animal origin, in particular ofporcine origin is used for preparation of the inventive composition.

According to the present invention at least 40% of the oxygen bindingsites of the oxygen carrier are charged with a non-O₂ ligand. Preferablyat least 50%, preferably at least 60%, more preferred at least 70%, evenmore preferred at least 80%, particularly preferred at least 90% or 95%of the hemoglobin or myoglobin is provided in ligand-charged form. Thischarge may already be applied during isolation of the carrier or afterits further purification, however, it is particularly preferred to carryout the isolation of the oxygen carrier in its protected form, whichmeans that during isolation or purification the ligand is providedto/contacted with the oxygen carrier.

The non-O₂ ligand preferably is carbon monoxide (CO) or nitrogenmonoxide (NO) or a mixture thereof. Both ligands have a high affinityfor the hemoglobin/myoglobin O₂ binding site(s) and serve as a protectoragainst oxidation of the central Fe³⁺ Ion of the heme.

The charged oxygen carrier(s) is/are preferably dissolved in an aqueousor organic medium, wherein an aqueous solution is preferred, in anamount of 0.1 to 35 wt.-%, preferably 0.1 to 20 wt.-%, more preferred0.1 to 15 wt.-%, to be ready for application.

The composition according to the present invention further comprises atleast one further additive, preferably selected from the groupcomprising electrolyte(s), stabilizer(s), anti-flocculant(s),preservative(s), pH buffering agent(s), solvent(s), antioxidant(s) andfilm-forming agent(s), more preferred selected from electrolyte(s),stabilizer(s), anti-flocculant(s), preservative(s) and pH bufferingagent(s). Most preferred the composition is in form of a solution andcomprises at least an electrolyte and optionally a stabilizer.

The solution may comprise physiologically compatible electrolytes, suchas salts, in suitable or desired amounts. The electrolytes may bepresent in amounts of 0.1 to 30 wt.-%, preferably 0.1 to 10%, butpreferably are present in a physiological concentration, respectively.Preferably the composition comprises a salt in the before mentionedamounts, like e.g. NaCl, KCl, NH₄Cl, CaCO₃, Na₂CO₃, K₂CO₃, MgSO₄,Na₂SO₄, CaCl₂, MgCl₂, sodium citrate, sodium lactate or mixtures of thementioned or similar without being restricted to these examples. Themost preferred salt is NaCl, particularly in a concentration of 0.9%(isotonic solution).

According to the invention it is particularly preferred that thecomposition comprises a compound acting as a stabilizer and/oranti-flocculant for proteins in particular for the hemoglobin/myoglobin,such as N-acetyl cysteine, cysteine, N-actyl methionine, methionine,non-chaotropic salts, polyols, like sugars, preferably disaccharides,and amino acids preferably each in amounts of 0.001 wt.-% to 20 wt.-%.

The polyols which may be employed are preferably low molecular weightpolyols although polymeric derivatives may be employed. Such polyolsinclude ethylene glycol, glycerol, erythritol and mannitol. Cyclicpolyols which may be employed incorporate one or more alicyclic ringsand may have at least one side chain. Preferred polyols includedisaccharides and sugar alcohols, for example lactitol, sorbitol andinositol. Compounds having 2 to 10 hydroxyl groups are preferred. Theamount of the polyol may be in the preferred range 0.001 to 20% morepreferably 1 to 15% most preferably 2 to 10% w/v.

Further the protein stabilizer additive may be selected from atris(hydroxymethyl)methyl compound of formula 1; (HOCH₂)₃C—R (1) whereinR is: C₁-C₄ alkyl, substituted C₁-C₄ alkyl, NH₂, NHC(CH₂OH)₃, C₁-C₄hydroxyalkyl; C₁-C₄ alkyl carboxylate, NR¹R² (wherein R¹ and R² may beindependently: H, C₁-C₄ alkyl, C₁-C₄ alkyl sulphonate, C₁-C₄hydroxyalkyl sulphonate). Examples of preferred compounds of formula (1)include: tris(hydroxymethyl)ethane; 1,1′,1″-tris(hydroxymethyl)propane;tris(hydroxymethyl)aminomethane or salts thereof for example chloride,maleate, phosphate, succinate salts; 1,3bis[tris(hydroxymethyl)methylamino]propane;bis(2hydroxyethyl)amino-tris(hydroxymethyl)methane; N[tris(hydroxymethyl)methyl]-2-aminoethane sulphonate; N[tris(hydroxymethyl)methyl]-3-aminopropane sulphonate; N[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropane sulphonate;N-[tris(hydroxymethyl)methyl]-glycine. Said compounds as well may beadded in the preferred range of 0.001 to 20% more preferably 1 to 15%most preferably 2 to 10% w/v.

Further the protein stabilizer additive may be selected from apolyelectrolyte. The polyelectrolyte may be a cationic or anionicpolyelectrolyte. Amphoteric polyelectrolytes may also be employed. Thecationic polyelectrolyte is preferably a polymer with cationic groupsdistributed along the molecular chain.

The cationic groups, which are preferably quaternary ammonium derivedfunctions, may be disposed in side groups pendant from the chain or maybe incorporated in it. Examples of cationic polyelectrolytes include:Copolymers of vinyl pyrollidone and quaternary methyl methacrylate egGafquat series (755N, 734, HS-100) obtained from ISP; substitutedpolyacrylamides; polyethyleneimine, polypropyleneimine and substitutedderivatives; polyamine homopolymers (Golchem CL118); polyamineco-polymers (eg condensates of epichlorohydrin and mono ordimethylamine); polydiallyl dimethyl ammonium chloride (polyDADMAC);substituted dextrans; modified guar gum (substituted withhydroxypropyltrimonium chloride); substituted proteins (eg quaternarygroups substituted on soya protein and hydrolysed collagen); polyaminoacids (eg polylysine); low molecular weight polyamino compounds (egspermine and spermidine). Natural or artificial polymers may beemployed.

Cationic polyelectrolytes with MW 150 to 5,000,000, preferably 5000 to500,000, more preferably 5000 to 100,000 may be employed. An amount of0.01 to 10% is preferred, more preferably 0.1 to 2%, especially 0.05 to5% w/v.

The anionic polyelectrolyte is preferably a polymer with anionic groupsdistributed along the molecular chain. The anionic groups, which mayinclude carboxylate, sulphonate, sulphate or other negatively chargedionisable groupings, may be disposed upon groups pendant from the chainor bonded directly to the polymer backbone. Natural or artificialpolymers may be employed.

Examples of anionic polyelectrolytes include: Gantrez (Sseries,AN-series); alginic acid and salts; carboxymethyl celluloses and salts;substituted polyacrylamides (e.g. substituted with carboxylic acidgroups); polyacrylic acids and salts; polystyrene sulphonic acids andsalts; dextran sulphates; substituted saccharides eg sucroseoctosulphate; heparin. Anionic polyelectrolytes with MW of 150 to5,000,000 may be used, preferably 5000 to 500,000, more preferably 5000to 100,000. An amount of 0.01% to 10% is preferred especially 0.05 to 5%more especially 0.1 to 2% w/v.

A particular preferred stabilizer is N-acetyl cysteine in an amount of 0to 10%, preferably 0.01 to 5%.

Further the composition may contain any preservative like e.g.phenoxyethanol, isothiazoline, sorbic acid or any other suitablepreservative known to skilled persons.

The composition may further preferably comprise any buffering agent. Allof the commonly known buffering agents may be used, like Tris/HCl,K₂HPO₄/KH₂PO₄, Na₂HPO₄/NaH₂PO₄, MOPS (3-(N-morpholino)propanesulfonicacid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS(3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Bicine(N,N-bis(2-hydroxyethyl)glycine), Tricine(N-tris(hydroxymethyl)methylglycine), TES(2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), SSC (saline sodiumcitrate), MES (2-(N-morpholino)ethanesulfonic acid) without beinglimited to these.

Suitable solvents in the composition according to the invention arepreferably water or aqueous solutions, organic solvents like alcohol,preferably ethanol, or polyethyleneglycol (PEG). Mixtures of saidsolvents as well can be used. Further natural oils may be used as asolvent for some of the ingredients and the composition may be providedas an emulsion. A particularly preferred solvent is water or an aqueoussolution.

Antioxidants useful for the present composition may be e.g. vitamin C,vitamin E, flavonoids, carotinoids, or salts or derivatives thereof.

Preferred film-forming agents are such agents commonly used in cosmeticapplication, like e.g. Acrylamide/Sodium, Acrylate Copolymer,Acrylates/Acrylamide Copolymer, Butyl Ester of PVM/MA Copolymer,Carboxymethyl Chitin, Chitosan, Hydroxypropyl Cellulose,Polyquaternium-36, PVP, PVP/VA Copolymer, VA/Crotonates Copolymer orVinyl Caprolactam/PCP/Dimethylaminoctyl Methylacrylate Copolymer.

All the above mentioned additives may be present in an amount of 0 to20, preferably 0.001, 0.01, 0.05 or 0.1 to 10, more preferably 0.5 to 5%(w/v), if not otherwise stated above.

If desired, further additives may be present, in particular in an amountof 0 to 20, preferably 0.1 to 20, preferably 0.2 to 15, particularly 0.5to 10 wt.-%. Preferred additives are nutrients for cells. They can beselected from glucose, e.g. in amounts of 0.1 to 5 wt.-%, insulin inamounts of up to 25 IU/ml, the natural amino acids, in particularcysteine, e.g. 0.1 to 5 wt.-%, or tissue factors, such as interleukinsin physiological amounts, up to a 10-fold amount thereof.

Preferably the composition represents an aqueous solution comprising

-   -   (a) an oxygen carrier, preferably hemoglobin or myoglobin,        wherein in at least 40% of said oxygen carrier the oxygen        binding site is charged by a non-O₂ ligand, and    -   (b) at least one further ingredient, selected from        electrolyte(s) preservative(s), stabilizer(s),        anti-flocculant(s), anticoagulant(s), pH buffering agent(s),        antioxidant(s), organic solvent, film-forming agent(s) and        crosslinking agent(s).

In a preferred embodiment of the present invention the compositioncomprises an oxygen carrier, which is isolated from whole blood of ahuman or animal, preferably from pigs.

Accordingly it is a further aspect of the invention to provide a processfor the purification of an oxygen carrier usable for the composition ofthe present invention from whole blood.

For preparation of the composition in the preferred embodiment theoxygen carrier is isolated from blood of a human or animal and isfurther purified to be essentially free of plasma and cellular membraneconstituents.

With “essentially free” is meant that the considered compound orcomposition doesn't comprise more than 20%, preferably not more than10%, even more preferred not more than 5% and particularly preferred notmore than 2% or less than 1% of the mentioned undesired compound(s).

The purification can comprise any suitable means or method steps, likee.g. selective lysis or precipitation, centrifugation,ultracentrifugation, fractionated centrifugation, chromatography methodslike anion exchange chromatography, size exclusion chromatography,affinity or adsorption chromatography, gel filtration or molecular sievechromatography, or dialysis, without being restricted to these examples,as far as by the applied methods the oxygen carrier is denaturated asless as possible. Preferably during isolation and purification theoxygen carrier remains essentially in solution.

When the oxygen carrier is isolated from whole blood, it is preferredthat either the cells comprising the oxygen carrier are separated fromother blood components or said cells are selectively lysed to deliverthe (soluble) oxygen carrier into solution and thereafter thenon-soluble components are separated. A combination of the two methodsas well is suitable. The lysis of the oxygen carrier containing cellsmay be carried out by any suitable lysis method, e.g. chemical lysis,osmotical lysis, mechanical lysis, thermal lysis or similar.

Cell debris may be separated by any suitable means or method. Thisincludes e.g. centrifugation, filtration, sedimentation and decantation,dialysis or any similar method.

For separating non-lysed cells or the cell debris from the solved oxygencarrier a common method is pelletation of the solid material. Forexample a centrifugation step may be carried out. Centrifugation with 2to 5000×g usually is sufficient for pelleting cells and cell debris.

For pelleting further non-solved components, e.g. any precipitatedeveloped during the purification process, at any time during thepurification process further centrifugation steps may be carried out, inparticular centrifugation steps using higher forces, up toultracentrifugation with up to 10⁶×g.

The purification of the oxygen carrier containing solution additionallyor as an alternative to any centrifugation step may comprise at leastone filtration step, preferably at least two, three or more filtrationsteps. This can be carried out either by using at least one, preferablyat least two, more preferably at least three filters (if more than onefilter is used in the present application we use the term “filtercascade”), or by one, two, three or more separate filtration steps.

Said filter cascade or the different filtering steps may include two,three, four, five ore more filters of different type, different materialand or different pore sizes. Further a deep bed filter like e.g. glasswool or similar may be used, preferably as a first filter material toretain coarse cell debris. If more than one filter is used, it ispreferred to use filters providing different pore diameters in decreasedorder. For example, if three different filters are used, the firstfilter (after the deep bed filter) may have an average pore size of 1 to0.5 μm, the second filter may have a pore size of 0.7 to 0.3 μm and thethird a pore size of 0.4 to 0.1 μm, wherein independent from theoverlapping ranges cited before the following filter in any case has asmaller pore size than the filter prior to that. By said filteringstep(s) solid and precipitated material having a larger size than thepore size of the used filters is essentially removed.

Further an ultrafiltration step may be included in the purificationprocess for purifying the oxygen carrier(s). By such an ultrafiltrationstep non-desired solved macromolecules can be separated. Preferably thesize exclusion limit is selected to separate macromolecules which arebigger (larger, higher molecular weight) than the desired oxygencarriers, accordingly said macromolecules are retained by the filter.Due to the molecular weight of hemoglobin of about 64,000 Dalton thesize exclusion limit of the ultrafiltration filter should be higher. Tomake sure that the yield of hemoglobin is not decreased by theultrafiltration step, it is preferred to select the size exclusion ofthe filter at about 100,000 Dalton, preferably at about 200,000 Dalton,more preferred at about 300,000 Dalton without being restricted to thesevalues.

Additionally or as an alternative any suitable chromatography step canbe carried out. A particularly preferred type of chromatography is ionexchange or size exclusion chromatography.

The same result may be obtained by a dialysis step using a dialysismembrane providing the above mentioned size exclusion limits, allowingthe oxygen carrier to pass, but retaining the macromolecules having ahigher molecular size.

To lower the amount of small molecular weight compounds in solution anadditional dialysis step may be carried out using a dialysis membranehaving a size exclusion limit of about 50,000 Dalton, allowing smallermolecules to pass, but retaining the oxygen carrier.

To diminish the virus and/or microorganism contamination in thecomposition it is particularly preferred to include a step of viruscontent degradation in the purification process. The virus content isreduced by this step, preferably to a burden of less than 10, preferablyless than 5, more preferably less than 2 virus particles per ml, andeven more preferred to 0. In this step it is preferred that the solutioncomprising the oxygen carrier is passed through a virus contentdegradation filter (“virus filter”). Such filters are commonly known andavailable on the market. Examples are Sartorius Virosart® CPV, Planova®15N,20N, Millipore Viresolve® NFP or PALL Pegasus® Grade LV6, withoutbeing limited to these. Alternatively or additionally, preferably afterthe passage through the filter, a treatment with UV light, in particularUV light of a wavelength of 245 nm may be applied to dispatch anyremaining viruses.

Optionally at any stage during the process of isolation of the oxygencarrier at least one heating step may be carried out. This stepcomprises the heating of the oxygen carrier containing suspension orsolution during the isolation procedure to a temperature in the range of40 to 85° C., preferably 60 to 80° C., more preferred in the range of 65to 75° C. The heating step is carried out preferably for 10 min to 6hours, preferably for 20 min to 4 hours and most preferred for 30 min to3 hours and may comprise several different temperatures within thebefore mentioned range.

According to the process of the present invention it is preferred thatthe oxygen carrier remains in solution during the whole purificationprocess. Further it is preferred that the oxygen carrier remains insolution during the whole purification process and during preparation ofthe composition of the present invention. This means that it ispreferred that the oxygen carrier is not precipitated in the process ofthe present invention and accordingly remains in its naturalthree-dimensional structure as present in its natural environment.

In a particular preferred process according to the present invention theprocess for purifying an oxygen carrier from whole blood comprises atleast the steps:

-   -   (a) separating plasma of the whole blood    -   (b) lysing the red blood cells    -   (c) charging the oxygen carriers with the ligand    -   (d) heating the sample to a temperature in the range of 40 to        85° C.    -   (e) separating the oxygen carrier from any non-desired blood        components.

By these steps an oxygen carrier containing solution is obtainable whichcan be used for the preparation of the composition of the presentinvention. In particular the oxygen carrier containing solutionobtainable by these steps may be concentrated to a desired amount of theoxygen carrier and to this solution the at least one furtheringredient(s) is/are added to obtain the composition of the presentinvention.

Step (a) of the present method can be carried out by any of the commonlyused methods for separating plasma from whole blood, preferably bycentrifugation or filtration. By centrifugation for about 30 min atabout 2000 to 5000 rpm, e.g. 4000 rpm red blood cells are pelleted,whereas soluble compounds and white blood cells remain predominantly inthe supernatant. By repeating resuspension and pelleting of the redblood cells e.g. 2 to 5 times, separation of the red blood cells fromthe undesired blood compounds can be increased.

Step (b) is preferably carried out by adding water, preferably distilledwater or a suitable sub-isotonic buffer, preferably a phosphate buffer,to the thickened blood of step (a). After lysing the red blood cellswith water or a sub-isotonic buffer preferably a salt is added to thesolution/suspension to obtain physiological concentration of said saltin solution. Preferably NaCl is added to an amount of 0.9% in solution.

Step (c) may be carried out after step (a), after step (b), after step(d) or after step (e), but is preferably carried out at least after step(b). It is particularly pointed out that step (c) is not necessarilycarried out immediately as a next step after step (b), but as well canbe carried out or repeated after step (d), after step (e) or anyfollowing treatment steps. The charging of the oxygen carrier in thesolution/suspension may be carried out by introducing gas in thesolution/suspension, preferably CO or NO gas or a mixture thereof. In apreferred embodiment CO gas is introduced into the solution/suspensionfor a time period long enough to obtain a >90% saturation in thesolution/suspension, preferably a >95% saturation.

Step (d) may be carried out after step (a), after step (b), after step(c) or after step (e), but is preferably carried out after step (c).Further the heating can be repeated during the isolation procedure. Thisstep comprises the heating of the oxygen carrier containing suspensionor solution during the isolation procedure to a temperature in the rangeof 40 to 85° C., preferably 60 to 80° C., more preferred in the range of65 to 75° C. The heating step is carried out preferably for 10 min to 6hours, preferably for 20 min to 4 hours and most preferred for 30 min to3 hours and may comprise several different temperatures within thebefore mentioned range.

In step (e) the oxygen carrier is purified from further non-desiredingredients still contained in solution, like non-lysed cells, celldebris, any precipitate or other non-soluble ingredients. Further theoxygen-carrier may be further purified by separating at least partiallynon-desired soluble compounds, like e.g. soluble macromolecules orsoluble compounds having low molecular weight.

Accordingly said step (e) may include several single steps, likefiltration, ultrafiltration, centrifugation, ultracentrifugation,chromatography, dialysis using different types of dialysis membranesproviding different size exclusion limits, washing steps, concentrationof the oxygen carrier content etc. Any of the methods cited above may beincluded in this purification step.

Preferably at least one centrifugation and/or at least one filtrationstep is comprised in step (e). E.g. the lysate may be spun in acentrifuge to separate remaining cells and cell debris or it is filterede.g. by a filter cascade as described above. The lysate can be as wellfirst centrifuged and thereafter filtered, or it may be filtered in afirst step through a deep bed filter and thereafter through at least onefilter or a filter cascade. By the centrifugation or the deep bed filterthe handling during any following filtering steps is simplified due toless material settling on and clogging the filter(s). If not a filtercascade is used, it is preferred that at least one filter is usedallowing to retain essentially all of the solid materials contained inthe suspension and allowing to pass all the solved components. In a morepreferred embodiment at least one of the used filter(s) is able toretain as well microorganisms, acting as a sterile filter. Furtherpreferred an ultrafiltration step and/or a step for diminishing thevirus and/or microorganism content of the solution can be carried out.Accordingly it is preferred that after step (e) the oxygen carriercontaining solution is essentially free of any non-solved particles,flocks or precipitate.

In step (e) additionally to any of the steps/methods cited above thesolution comprising the desired oxygen carrier may be washed and/orconcentrated. By “washing” is meant that molecules smaller than thedesired oxygen carrier (having lower molecular weight) are separated,preferably by adding the same or a multifold (e.g. 5 to 10 fold) amountof an isotonic solution to the oxygen carrier containing solution andthereafter filtering the obtained (diluted) solution by a filterretaining the oxygen carrier and allowing smaller molecules to pass. Forwashing the solution preferably a 0.9% NaCl solution is used. Thewashing step may be repeated 2 or 3 or 4 or 5 or up to 10 times. Apreferred embodiment is exemplified by the use of a filter having a sizeexclusion limit of 5,000 Dalton, 10,000 Dalton or 20,000 Dalton,allowing smaller molecules to pass. In this step the oxygen carriercontaining solution (preferably after washing) may be concentrated to adesired concentration of the oxygen carrier, e.g. to a concentration of50 g/l, 100 g/l or 200 g/l without being restricted to these amounts.Any desired concentration can be obtained either by concentrating byfiltration or by adding 0.9% NaCl or a similar isotonic solution.

The so obtainable oxygen carrier containing solution can then be used toprepare the composition of the present invention by adding the at leastone further ingredient described above to the solution in the desiredamount.

In a preferred embodiment the composition of the present invention isprepared by adding to the oxygen carrier containing solution at least apreservative, preferably a pharmaceutically acceptable preservative likee.g. phenoxyethanol, parabenes, sodium benzoate, benzyl alcohol,hexachlorophen and an antioxidant and/or stabilizer like e.g.N-acetylcysteine, sodium octanoate, N-acetyl-1-tryptophanate,N-acetyl-methioninate, vitamin E, vitamin C, methyl prednisolone ormannitol. Additionally any of the further ingredients described abovemay be added additionally.

The finished composition may be sterilized again, if desired, e.g. byheating, filtration, centrifugation, addition of preservatives, vapourapplication, gas application or UV-application or a combination of atleast two of them, preferably by a further sterile filtration step andis preferably filled in sterile containers or sterile bags for storing.

According to a preferred embodiment the sterile bags are positioned inan aerosol can for later use. One example can be a Bag-on-Valve system,comprising a bag, e.g. a laminated aluminium bag and an aluminium or tinplate aerosol can. Due to the separation of product and propellant,Bag-on-Valve can be used with compressed air or nitrogen at a pressuree.g. from 2 to 9 bar.

According to the invention, the composition is preferably used for thetreatment of open wounds, preferably chronic wounds of humans andanimals, wherein the use for the treatment of humans is preferred. Thepreferred use is by topical treatment of the wound(s) with the chargedoxygen carrier(s) described.

Surprisingly it was found that it is possible to effectively treat openwounds with said charged oxygen carriers. As described above the healingof chronic wounds is often diminished due to the decreased oxygensupplement. It appears therefore logic that the treatment of the woundsrequires the oxygen-charged oxygen carriers for treatment. However,according to the present invention it was found that it is possible toobtain a positive external wound healing by applying an oxygen-carriercharged with CO or NO. Without being bound our theory is that due to theoxygen partial pressure in air the ligand may be replaced by oxygen whenapplied to the wound and accordingly the wound healing is supported.

The composition of the present invention has the clear advantage ofincreased stability of the oxygen-carrier due to its non-oxidized statusbased on the charge with the CO or NO ligand resulting in a goodpreparation and storing stability. Further the composition is easy toapply and good to handle and provides a safe and effective approach toimprove wound healing by facilitated diffusion mediated by the oxygencarrier/agent.

The composition of the present invention is applied externally.Depending on the state of the wound, it is applied on, preferablysprayed on the wound area in form of a fine spray.

According to the invention, it has been shown that open wounds,particularly also chronic wounds having very different causes, can beeffectively treated. These can be wounds after operations, after trauma,after injuries, wounds with poor healing or hypoxic wounds, or alsowounds caused by degenerative changes in the tissue. In this connection,they can be wounds caused by degenerative changes of the arterial bloodvessels and wounds resulting from chronic venous insufficiency. Theseparticularly include decubitus as well as chronic wounds, particularlythose resulting from diabetes. Further wounds caused by burn (either byheat, by chemicals or by freezing) or by scalding can be effectivelytreated.

FIGURES

FIG. 1 shows the O₂ saturation curve of hemoglobin dependent from the O₂surrounding partial pressure.

EXAMPLES Example 1

Hemoglobin was isolated from whole blood of pigs by separating the redblood cells from serum, lysing the collected red blood cells, pelletingcell debris, charging the hemoglobin with CO by introducing CO gas untilsaturation of the liquid sample is obtained, heating the solution,carrying out several filtration steps, including a virus filtration stepand washing the obtained hemoglobin solution by adding twice a 2-foldvolume of 0.9% saline and filtering the solution.

A ready-to-use composition for wound treatment was prepared, comprising10% of purified and stabilized hemoglobin, 0.05% N-acetyl cysteine and0.7% phenoxy ethanol in 0.9% NaCl. The composition was charged againwith CO gas, separated into 10 portions and packaged into an aerosolcan, respectively.

Example 2

A first portion of the composition of Example 1 was used immediatelyafter preparation to treat a female patient (Diagnosis: Diabetes,Hypertension) with a chronic wound at the right foot (7×2 cm). The woundwas treated once daily by spraying the composition onto the wound. After3 weeks first signs of healing were visible. The next weeks the healingprocess was pronounced and complete closing of the wound was obtainedafter 18 weeks.

Example 3

A second portion of the composition of Example 1 was stored at 4° C. for2 years after preparation and thereafter used to treat a chronical woundof a male patient suffering from diabetes mellitus type II. Thecomposition was applied to a chronic superficial ulcer (4×3cm) at theleft tibia of the patient. The wound was treated once daily. After twoweeks a healing was appearing, after 4 weeks the wound was healed morethan 50% of the baseline. The next weeks a fast healing was observed andcomplete closing of the wound was obtained after 8 weeks.

Example 4

A third portion of the composition of Example 1 was stored at 10° C. for2 years. Thereafter it was used for treatment of a chronic superficialulcer (4×2 cm) at the right leg of a male patient suffering fromdiabetes mellitus type II. The wound was treated once daily. After 2.5weeks healing was visible. The next weeks a fast healing was observedand complete closing of the wound was obtained after 12 weeks.

Example 5

A portion of the composition of Example 1 was used to treat a malepatient after contact with hot boiling water and steam. The diagnosiswas 1^(st) and 2^(nd) degree burns on his face.

Initial treatment with cool gel and Neosporin for one week showed noimprovement and by the tending physician the application of skin graftson the nose and other parts of the face, like eyelids was proposed.

Instead the patient was treated with the composition of example 1 for 7weeks. The composition was applied to the skin by spraying three or fourtimes per day. A fast improvement and healing of the skin was observedand the patient was discharged from the hospital after seven weeks. Noskin grafts were required.

Example 6 Comparison of Charged/Non-Charged Hemoglobin

A) A charged hemoglobin-spray was prepared according to Example 1

A ready-to-use composition for wound treatment was prepared, comprising10% of purified and stabilized hemoglobin, 0.05% N-acetyl cysteine and0.7% phenoxy ethanol in 0.9% NaCl. The composition was charged againwith CO gas, separated into 20 portions and packaged into an aerosolcan, respectively.

The composition can be stored between 4° C. and room temperature formonths to years.

B) A second portion of the pig whole blood was treated as in Example 1with the exception that no CO charging was carried out. During theheating step a considerable amount of hemoglobin precipitated. Thefollowing filtration steps were difficult to carry out. The yield ofpurified hemoglobin per liter whole blood decreased to less than 30% ofthe yield when the CO charging is carried out before heating. This showsthat charging the samples with CO during preparation stabilizes thehemoglobin in a way that during a heating step considerably lesshemoglobin precipitates.

A ready-to-use composition for wound treatment was prepared, comprising10% of purified hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxyethanol in 0.9% NaCl. The composition was portioned and packaged intoaerosol cans, respectively.

C) A further portion of hemoglobin was isolated from whole blood of pigsby a method as described in Example 1 of WO2003/077941 without chargingthe hemoglobin during preparation: the hemoglobin was freed from plasmaand cellular membrane constituents without heating by means ofcentrifugation and ultrafiltration, and was purified.

A ready-to-use composition for wound treatment was prepared, comprising10% of purified hemoglobin, 0.05% N-acetyl cysteine and 0.7% phenoxyethanol in 0.9% NaCl. The composition was separated into 10 portions andpackaged into an aerosol can, respectively.

A such prepared non-charged hemoglobin spray was freshly prepared duringthe treatment period as often as needed (all three days) and stored at4° C. maximal for one day before use.

Treatment of Patients:

Several male patients (age 65-75) with Diabetic foot ulcer at the lowerleg were treated for three months with hemoglobin spray according to A)(stored at 10° C. for up to 6 months) or a freshly prepared hemoglobinspray according to C) (storage at 4° C. for 3 days max., i.e. all threedays a fresh composition was used) until complete wound healing wasobtained at all of the patients. According to the attendingdermatologists, the wound healing obtained with the compositionsaccording to A) showed in all cases less secretion, less incrustationand/or less suppuration in comparison to wounds treated by thehemoglobin spray according to C) and in particular in comparison towounds treated conventionally. Due to the minor problems the woundstreated with the charged hemoglobin composition according to A) showed afaster healing per cm² than the wounds treated with the non-chargedhemoglobin spray C) and much faster than the conventionally treatedwounds. The patients treated with composition A) furthermore reportedless discomfort .

Example 7

Samples of compositions prepared according to Example 6A), Example 6B)or Example 6C), respectively, were examined considering the O₂ charge ofthe samples as well as the production of methemoglobin, a species whichis unable to bind O₂. Furthermore methemoglobin affects hemoglobinmolecules in the immediate vicinity in a way that these can still bindO₂, but cannot release it no more.

All the samples of Table 1 were diluted 1:1 with a 0.9% NaCl solution toimitate the conditions of a wound treatment. Samples 7.1 to 7.8 weremeasured immediately after dilution (storing of samples 7.5 to 7.8 wasat room temperature before dilution). Gas treatment was as follows: 6 mlof said diluted solution were transferred into a 30 ml glass flask each.Gas treatment was carried out by filling the glass flask completely withthe respective gas, closing the flask and pivoting the sample for 30sec. All the samples were stored for the mentioned time period at 30° C.The samples were treated according to the conditions described in Table1 and the total amount of hemoglobin (hemoglobin+methemoglobin), theoxygen content, the methemoglobin content and optionally the CO contentwas measured.

TABLE 1 totalHb O₂ CO MetHb Example sample treatment g/dL % % % 7.1Freshly prepared comp. none 5.5 1.7 96.3 3.9 according to Example 6A)7.2 Freshly prepared comp. none 5.7 26.8 5.1 According to Example 6B)7.3 Freshly prepared comp. none 5.6 27.0 4.7 According to Example 6C)7.4 Composition of Example 6A), none 5.9 1.8 96.5 3.8 stored for 3 month(10° C.) 7.5 Composition of Example 7.2 Stored for 24 h 5.6 26.9 11.27.6 Composition of Example 7.2 Stored for 48 h 5.5 27.0 17.0 7.7Composition of Example 7.3 Stored for 24 h 5.7 26.5 10.5 7.8 Compositionof Example 7.3 Stored for 48 h 5.6 26.7 16.2 7.9 Composition of Example7.4 0.5 h O₂ 5.8 13.7 84.7 4.4 7.10 Composition of Example 7.4 1 h O₂5.7 19.6 79.1 4.2 7.11 Composition of Example 7.4 2 h O₂ 5.8 25.7 73.14.3 7.12 Composition of Example 7.4 3 h O₂ 5.9 30.7 68.1 4.2 7.13Composition of Example 7.4 24 h O₂ 5.7 33.5 59.6 9.8 7.14 Composition ofExample 7.4 48 h O₂ 5.8 32.5 55.9 15.5 7.15 Composition of Example 7.472 h O₂ 5.7 31.7 55.8 16.0 7.16 Composition of Example 7.4 0.5 h CO₂ 5.84.0 91.8 5.7 7.17 Composition of Example 7.4 1 h CO₂ 5.7 6.0 90.0 5.77.18 Composition of Example 7.4 2 h CO₂ 5.9 9.2 86.7 5.8 7.19Composition of Example 7.4 3 h CO₂ 5.7 9.6 85.1 6.9 7.20 Composition ofExample 7.4 24 h CO₂ 5.6 4.9 77.5 19.7 7.21 Composition of Example 7.448 h CO₂ 5.8 8.2 68.2 27.4 7.22 Composition of Example 7.4 72 h CO₂ 5.79.4 65.9 28.3 7.23 Composition of Example 7.4 0.5 h air 5.8 9.2 89.0 4.57.24 Composition of Example 7.4 1 h air 5.7 12.4 85.9 4.4 7.25Composition of Example 7.4 2 h air 5.9 18.0 80.5 4.3 7.26 Composition ofExample 7.4 3 h air 5.6 20.7 77.9 4.3 7.27 Composition of Example 7.4 24h air 5.8 24.3 69.3 9.1 7.28 Composition of Example 7.4 48 h air 5.725.4 64.3 13.9 7.29 Composition of Example 7.4 72 h air 5.7 26.8 62.214.3

As can be seen from the results in Table 1 the compositions of theinvention, wherein the oxygen carrier is charged with CO, not only canbe stored for a long time without forming methemoglobin, but further areable to replace the bound CO by O₂ when it is offered to the chargedoxygen carrier. If exposed to 100% O₂ (examples 7.9 to 7.15) the O₂saturation of the hemoglobin increases very fast.

If the composition is exposed to CO₂, representing the situation insideof mammalian pathway-active tissue, an increased amount of methemoglobinis formed (examples 7.16 to 7.22).

The O₂ partial pressure in air is only about 21%, thus according to theO₂ saturation curve of hemoglobin shown in FIG. 1, the maximum possibleO₂ saturation of hemoglobin with oxygen under air is about 29%.Considering Examples 7.23 to 7.29 the surprising result is that within 3hours the composition exposed to air is charged with 20.7% oxygen, butstill has a very low methemoglobin content. When external wounds aretreated, the composition is sprayed to the (cleaned) wound surface andremains in contact with air.

These results show that a composition according to the inventioncomprises a stabilized oxygen carrier which after several months ofstoring provides high oxygen transport when it is in contact with air.

1.-16. (canceled)
 17. A method for the external treatment of an openwound surface, comprising spraying on the open wound surface an aqueouscomposition comprising: (a) an oxygen carrier, wherein at least 40% ofoxygen binding sites of said oxygen carrier are charged by a non-O₂ligand, such that the oxygen carrier is sprayed on the open woundsurface with the oxygen carrier in its non-oxidized state in the sprayand provides availability of oxygen to the open wound surface in thepresence of oxygen partial pressure in air as opposed to a pressurizedchamber of pure oxygen, and (b) at least one further ingredient selectedfrom the group consisting of electrolyte(s), preservative(s),stabilizer(s), anti-flocculant(s), anticoagulant(s), pH bufferingagent(s), solvent(s), antioxidant(s), film-forming agent(s) andcrosslinking agent(s), wherein the oxygen carrier is hemoglobin and thenon-O₂ ligand is nitrogen monoxide (NO).
 18. The method according toclaim 17, wherein the wounds to be treated are selected from the groupconsisting of chronic wounds, operation wounds, injury wounds, woundsafter trauma, open wounds, wounds with poor healing, hypoxic wounds,wounds arising from degeneration or stenosis of arterial blood vessels,diabetic wounds, chronic venous insufficiency wounds, decubitus ulcerwounds, burn wounds, chemical burns, freezing burns, and scaldingwounds.
 19. The method according to claim 17, wherein the compositionadministered is an organic solution.
 20. The method according to claim17, wherein at least 50% of the oxygen carrier is provided in non-O₂ligand-charged form.
 21. The method according to claim 20, wherein atleast 60% of the oxygen carrier is provided in non-O₂ ligand-chargedform.
 22. The method according to claim 21, wherein at least 70% of theoxygen carrier is provided in non-O₂ ligand-charged form.
 23. The methodaccording to claim 22, wherein at least 80% of the oxygen carrier isprovided in non-O₂ ligand-charged form.
 24. The method according toclaim 23, wherein at least 90% of the oxygen carrier is provided innon-O₂ ligand-charged form.
 25. The method according to claim 17,wherein the oxygen carrier is naturally occurring hemoglobin of human oranimal origin.
 26. The method according to claim 17, wherein the oxygencarrier is artificially-treated, crosslinked hemoglobin.
 27. The methodaccording to claim 17, wherein the composition administered is providedin sterilized form.
 28. The method according to claim 17, wherein thecomposition administered is provided in an aerosol can.
 29. The methodaccording to claim 28, wherein the composition administered is providedin a pressure pack aerosol can.
 30. The method of claim 17, wherein theligand is replaced by oxygen when the spray is applied to the open woundsurface due to oxygen partial pressure in the air.
 31. A method for theexternal treatment of an open wound surface, comprising spraying from anaerosol on the open wound surface an aqueous composition comprising: (a)a hemoglobin oxygen carrier, wherein at least 40% of oxygen bindingsites of the hemoglobin oxygen carrier are charged by nitrogen monoxideto render the hemoglobin oxygen carrier in a non-oxidized/non-activatedstatus, such that the oxygen carrier is sprayed on the open woundsurface with the oxygen carrier in its non-oxidized state in the sprayand provides availability of oxygen to the open wound surface in thepresence of oxygen partial pressure in air as opposed to a pressurizedchamber of pure oxygen, and (b) at least one further ingredient selectedfrom the group consisting of electrolyte(s), preservative(s),stabilizer(s), anti-flocculant(s), anticoagulant(s), pH bufferingagent(s), solvent(s), antioxidant(s), film-forming agent(s) andcrosslinking agent(s).
 32. The method of claim 17, wherein the non-O₂ligand is nitrogen monoxide (NO), carbon monoxide (CO), or a mixturethereof.
 33. The method of claim 31, wherein the non-O₂ ligand isnitrogen monoxide (NO), carbon monoxide (CO), or a mixture thereof.